We have identified the epitopes targeted by human monoclonal antibodies that react specifically with antigens associated with particular cancers. This invention also relates to diagnostic procedures, antibody screening and cancer therapy using these epitopes as targets and as active portions of immunogens.
Antibodies are protein molecules normally synthesized by B-cell lymphocytes produced by bone marrow and carried in the blood stream. For any antigen in the body, i.e., any foreign or non-self self molecule, from a simple organic chemical to a complex protein, antibodies are produced that recognize and attach to that particular chemical structure. The unique chemical structure on the antigen to which a particular antibody can bind is referred to as an antigenic determinant or epitope. B-cell lymphocytes in the body, referred to as B-cells, lymphocytes, or leukocytes, exist as hundreds of millions of different genetically programmed cells, each producing an antibody specific for a different epitope. An antigen, which stimulates antibody production, can have several epitopes on its surface. On encountering an antigen, a B-cell carrying on its surface an antibody specific for an epitope on that antigen will replicate. This clonal expansion results in many daughter cells that secrete that antibody into the blood stream.
Because of the specificity of antibodies in recognizing and binding to antigens, it was desired to identify antibodies that are specific for a single epitope, thus binding only to antigens or tissues having that particular epitope. Once the epitope was identified it was available for screening antibodies produced by cell lines, and even for immunopurifying antibodies from polyclonal serum. As part of a larger molecule, synthetic or natural, the epitope can also be used as an effective immunogen.
Monoclonal antibodies are synthesized in pure form uncontaminated by other immunoglobulins. With monoclonal antibody producing cells it is possible to produce virtually unlimited quantities of an antibody that is specific for one epitope on a particular antigen. Antibodies specific for particular cancer can be used in various methods of treatment and diagnosis.
Monoclonal antibodies increase the specificity of chemotherapeutic drugs, toxins and radioactive isotopes, thus increasing their efficacy while decreasing their toxicity by dramatically reducing the quantities required. In addition, antibodies conjugated with radionuclides or metallic tracers can be used for imaging for in vivo diagnosis and localization of metastases, such as with proton emission (PET), nuclear magnetic resonance (NMR), computed tomography (CT), and planar and single photon emission computed tomography, and for use in labeling tumor tissue to be identified during surgery using a probe. The antibodies can also be used for detecting the presence of tumor antigens in blood, as a diagnostic or prognostic test for cancer.
The existence of antigens associated with animal tumors was documented in the last century, and the antigenic character of human cancers has been well established, primarily through recent studies with monoclonal antibodies. However, until the research that resulted in this invention, few cancer antigens have actually been characterized in molecular terms and only one group of antigenic determinants associated with human cancers, immunoglobulin idiotypes of B-cell tumors, has been described as being uniquely tumor-specific, i.e., occurring with a high frequency on tumor cells and not occurring to any significant degree on normal tissues (Oldham and Smalley, J. Biol. Response Modifiers, 1983; Stratte et al, J. Biol. Response Modifiers, Volume 1, 1982).
Past attempts at deriving monoclonal antibodies specific for human cancers have taken two routes with respect to B-cells: 1) B-cells have been extracted from spleens of mice that were immunized against human tumors, U.S. Pat. No. 4,172,124; and 2) human B-cells have been extracted from either peripheral blood or from lymph nodes draining tumors in cancer patients. Neither approach has yielded satisfactory results.
Mice immunized against human tumors have too broad a reactivity. That is, most of the mouse monoclonal antibodies generated react with human antigens present on normal as well as on tumor tissue. An antibody that reacts only with tumor cells is very difficult to select from among the large variety of antibodies produced. For example, 20,000 hybridomas derived from mice immunized with human small-cell lung carcinoma were screened for reactivity with tumor cells (Science, 1982, 216:283), resulting in a very low frequency (&lt;0.4%) of reactivity observed by this research group. By contrast, the present invention results in up to 16% of the hybridomas derived from immunized colon patients produce monoclonal antibodies that react specifically with tumor cells. In addition, monoclonal antibodies derived from mouse B-cells have limited potential for application in cancer therapy. After repeated administration they stimulate the human immune system to produce "anti-mouse" antibodies which, in clinical trials, have been shown to neutralize the activity of mouse monoclonal antibodies. The use of our human monoclonal antibodies can circumvent these difficulties.
Another apparent difference between human and mouse monoclonal antibodies is their pattern of labeling. Previous studies with mouse antibodies have demonstrated that there is often a heterogenous labeling of cells within tumor sections. This pattern of reactivity has been attributed by some authors to antigenic heterogeneity of tumor cells (Hand et al., Cancer Research, 43:728-735, 1983). In contrast, the human monoclonal antibodies developed by our strategy were homogeneous in terms of their reactivity with tumors to which they did react. A plausible explanation for the heterogenous staining of mouse monoclonal antibodies is that it is a reflection of the murine immune recognition of phase- or cell-cycle-specific differentiation antigens abundant on the tumor cells rather than putative tumor associated antigens. It is not unreasonable to expect that when one immunizes mice with human tumor cells there would be substantial antigenic competition resulting in the more abundant and more predominant tissue-type and differentiation antigens successfully competing with proportionally fewer tumor associated antigens for immune responsiveness by the host. Thus, autologous immunization of man may result in the elicitation of antibodies against the group of antigens normally poorly immunogenic in mice. This evidence suggests that humans and mice may respond to different tumor antigens. In concert with this hypothesis is our finding that none of the first 36 human monoclonal antibodies we produced appeared to react with carcinoembryonic antigen (CEA), an antigen frequently recognized by murine monoclonal antibodies made against human tumor cells.
The major problem in creating monoclonal antibodies specific for human tumor antigens has been the inability to find a source of specifically immune B-cells (Science, 1982, 216:285). In humans, the initial foci of cancer cells tend to grow over long periods of time, from 1% to 10% of the human lifespan, before there is any palpable clinical evidence of the disease. By this time patients are immunologically hyporesponsive to their tumors, or possibly immunologically tolerant. Thus, prior to this work, the development of human monoclonal antibodies specific for tumor associated antigens and the identification of their target epitopes could not reproducibly be obtained.
Identification of the tumor specific epitopes of the present invention resulted from research beginning with efforts to develop monoclonal antibodies specifically reactive with tumor-associated antigens that induced an immune response in patients having particular cancers. This was a new and more effective approach for obtaining monoclonal antibodies using peripheral blood B-cells from patients immunized with cells from their own tumors in specific vaccine preparations. To achieve active specific immunotherapy, patients were immunized with cells from their own tumors. Humans mounting an objective immune response against tumor cells were specifically found to be a good source of activated B-cells. The peripheral blood of these patients, who had been actively immunized against their own tumors, was an abundant source of such activated B-cells.
Clinical studies demonstrated that an objective immune response is generated on treating patients having the particular cancer by skin testing, i.e., delayed cutaneous hypersensitivity (DCH). Immunized patients showed delayed cutaneous hypersensitivity to their own colorectal cancers. In addition, the monoclonal antibodies developed from the immunized patient's B-cells reacted with tumors of the same histological type in other patients. These results indicated that the patient's humoral immune response, production of antibodies, was directed against colorectal cancer antigens generally and was not unique to the immunized patient's own tumor. This general response, which indicated that there are specific antigens associated with tumors that contain unique epitopes, is especially important for the development of a standardized vaccine.
The generation of B-cells that produce antibodies having reactivity specific for particular epitopes on tumor cell associated antigens, particularly cell surface antigens as in the majority of cases, is an advantageous result that was speculative, at best, when the immunization studies were begun. So, too, was the discovery that these are particular epitopes that are presented by tumor cells, and are found to a comparatively small degree expressed in normal tissue. Only the immunization treatment was observed and measured during the animal studies on which the human immunization procedures were based, not the production of tumor specific antibodies.
The general immune response accompanied by an improvement in the subject's condition was indicative of a cellular response in which macrophages and T-cells become activated in the presence of tumor cell antigens and destroy the tumor cells. Although an antibody response would predictably be triggered by immunization under most circumstances, the time course of the antibody response and the cellular response would in most instances be different. Moreover, the fact that the patients were being immunized with autologous tumor cells, i.e., the patient's own tumor cells, and the experience of previous investigators that little or no antibody production is triggered by a patient's own tumor, made the discovery that B-cells that produce tumor specific antibodies are generated after immunization an unexpected beneficial result. We then discovered that there are tumor specific antigens having epitopes that are uniquely effective targets.
Some cellular and humoral immune responses can occur independently of each other. For example, it is possible to mount a humoral response in the absence of demonstrable cellular immunity. Conversely, potent cellular immunity, particularly delayed cutaneous hypersensitivity (DCH), may develop despite a minimal antibody response. It was surprising, therefore, for the subjects who showed a positive response to active immunotherapy to have been excellent sources of B-cells producing tumor specific antibodies, particularly to cell surface antigens.
The isolation of B-cells that produce tumor specific antibodies required the preparation of successful vaccines for active specific immunization, procedures for extracting immunized B-cells, the production of monoclonal antibody producing cell lines and the production of monoclonal antibodies. Malignant tumors were digested using enzyme preparations. The cells obtained were treated to yield a non-tumorigenic tumor cell preparation having the requisite cell viability, which was injected as a vaccine into the subject from which the tumor was obtained. Peripheral blood B-cells were obtained from the inoculated subject after a predetermined interval and were used to prepare monoclonal antibody producing cells by fusing with myeloma cells, after which the fused cells were screened for the synthesis of immunoglobulin. Monoclonal antibody producing cells were also obtained by selecting spontaneously transformed B-cells that were able to survive in continuous culture, and by exposing B-cells to an agent capable of transforming cells, such as Epstein Barr Virus (EBV) or another lymphotropic virus.
Larger amounts of antibodies were obtained by fusing EBV-transformed cells with mouse myeloma cells and human-mouse heteromyelomas. Cells that synthesized immunoglobulin were tested for production of antibodies that react with antigens characteristic of the malignant tissue. Those selected were cultured to produce monoclonal antibodies that react with the particular type of tumor with which the subject was afflicted. Our identification of novel epitopes reactive with these antibodies makes direct screening for other useful immunoglobulins possible. The monoclonal antibodies identified can be used as radioimmunoscintography (RIS) agents for diagnostic purposes and for carrying therapeutic agents to primary tumor and metastatic sites. The epitopes themselves are also directly useful for isolating antibodies directed against tumor antigens and as targets for in vivo imaging and therapy, and for in vitro tissue analysis. They may also be used, when combined with larger molecules, as immunogens for immunotherapy and for antibody production.
Identification of epitopes after raising monoclonal antibodies is not assured of success because antigen sequence information is normally not available, the antigens may not be simple proteins, and the epitopes themselves may be three dimension conformational epitopes with little or no reactivity in linear sequences. The present epitopes could only be identified and sequenced after our discovery that the 16.88 and 88BV59 antibodies reacted with certain cytokeratins for which sequence information was available.